Elaine Machado Benelli

Identification and characterization of the two-component NtrY/NtrX regulatory system in Azospirillum brasilense

Two Azospirillum brasilense open reading frames (ORFs) exhibited homology with the two-component NtrY/NtrX regulatory system from Azorhizobium caulinodans. These A. brasilense ORFs, located downstream to the nifR3ntrBC operon, were isolated, sequenced and characterized. The present study suggests that ORF1 and ORF2 correspond to the A. brasilense ntrY and ntrX genes, respectively. The amino acid sequences of A. brasilense NtrY and NtrX proteins showed high similarity to sensor/kinase and regulatory proteins, respectively. Analysis of lacZ transcriptional fusions by the beta-galactosidase assay in Escherichia coli ntrC mutants showed that the NtrY/NtrX proteins failed to activate transcription of the nifA promoter of A. brasilense. The ntrYX operon complemented a nifR3ntrBC deletion mutant of A. brasilense for nitrate-dependent growth, suggesting a possible cross-talk between the NtrY/X and NtrB/C sensor/regulator pairs. Our data support the existence of another two-component regulatory system in A. brasilense, the NtrY/NtrX system, probably involved in the regulation of nitrate assimilation.

Recent developments in the structural organization and regulation of nitrogen fixation genes in Herbaspirillum seropedicae

Herbaspirillum seropedicae is a nitrogen-fixing bacterium found in association with economically important gramineae. Regulation of nitrogen fixation involves the transcriptional activator NifA protein. The regulation of NifA protein and its truncated mutant proteins is described and compared with that of other nitrogen fixation bacteria. Nitrogen fixation control in H. seropedicae, of the beta-subgroup of Proteobacteria, has regulatory features in common with Klebsiella pneumoniae, of the gamma-subgroup, at the level of nifA expression and with rhizobia and Azospirillum brasilense, of the alpha-subgroup, at the level of control of NifA by oxygen.

Structural organization and regulation of the nif genes of Herbaspirillum seropedicae

Abstract The promoter regions of nifA and nifH genes of Herbaspirillom seropedicae have been isolated and investigated, and the nifA promoter has been characterized in detail. Both regions contain a −24 −12 type promoter and NifA-binding sites; the nifA promoter also has an NtrC-binding site. The nifA expression is activated by NtrC and repressed by ammonium ions but not by oxygen. When the NtrC-binding site was deleted nifA expression was NifA-dependent. The native NifA protein of H. seropedicae was unable to activate nifH expression in Escherichia coli whereas a truncated protein, lacking the N-terminal domain was able to activate nifH expression in the absence of oxygen and presence of iron, indicating that the N-terminal domain regulates NifA activity. The truncated protein also activates nifH in Azospirillum brasilense in the presence or absence of ammonium ions. This suggests that the N-terminal domain senses the nitrogen status of the cell.

The Streptococcus mutans GlnR protein exhibits an increased affinity for the glnRA operon promoter when bound to GlnK

The control of nitrogen metabolism in pathogenic Gram-positive bacteria has been studied in a variety of species and is involved with the expression of virulence factors. To date, no data have been reported regarding nitrogen metabolism in the odontopathogenic species Streptococcus mutans. GlnR, which controls nitrogen assimilation in the related bacterial species, Bacillus subtilis, was assessed in S. mutans for its DNA and protein binding activity. Electrophoretic mobility shift assay of the S. mutans GlnR protein indicated that GlnR binds to promoter regions of the glnRA and amtB-glnK operons. Cross-linking and pull-down assays demonstrated that GlnR interacts with GlnK, a signal transduction protein that coordinates the regulation of nitrogen metabolism. Upon formation of this stable complex, GlnK enhances the affinity of GlnR for the glnRA operon promoter. These results support an involvement of GlnR in transcriptional regulation of nitrogen metabolism-related genes and indicate that GlnK relays information regarding ammonium availability to GlnR.

Adsorption of protein glnB of Herbaspirillum seropedicae on Si(111) investigated by AFM and XPS

The protein GlnB-Hs (GlnB of Herbaspirillum seropedicae) in diazotroph micro-organisms signalizes levels of nitrogen, carbon, and energy for a series of proteins involved in the regulation of expression and control of the activity of nitrogenase complex that converts atmospheric nitrogen in ammonia, resulting in biological nitrogen fixation. Its structure has already been determined by X-ray diffraction, revealing a trimer of (36 kDa) with lateral cavities having hydrophilic boundaries. The interactions of GlnB-Hs with the well-known Si(111) surface were investigated for different incubation times, protein concentrations in initial solution, deposition conditions, and substrate initial state. The protein solution was deposited on Si(111) and dried under controlled conditions. An atomic force microscope operating in dynamic mode shows images of circular, linear, and more complex donut-shaped protein arrangement, and also filament types of organization, which vary from a few nanometers to micrometers. Apparently, the filament formation was favored because of protein surface polarity when in contact with the silicon surface, following some specific orientation. The spin-coating technique was successfully used to obtain more uniform surface covering.

ADEMO/D: An adaptive differential evolution for protein structure prediction problem

Abstract Protein Structure Prediction (PSP) is the process of determining three-dimensional structures of proteins based on their sequence of amino acids. PSP is of great importance to medicine and biotechnology, e.g., to novel enzymes and drugs design, and one of the most challenging problems in bioinformatics and theoretical chemistry. This paper models PSP as a multi-objective optimization problem and adopts ADEMO/D (Adaptive Differential Evolution for Multi-objective Problems based on Decomposition) on its optimizer platform. ADEMO/D has been previously applied to multi-objective optimization with a lot of success. It incorporates concepts of problem decomposition and mechanisms of mutation strategies adaptation. Decomposition-based multi-objective optimization tends to be more efficient than other techniques in complex problems. Adaptation is particularly important in bioinformatics because it can release practitioners, with a great expertise focused on the application, from tuning optimization algorithm’s parameters. ADEMO/D for PSP needs a decision maker and this work tests four different methods. Experiments consider off-lattice models and ab initio approaches for six real proteins. Results point ADEMO/D as a competitive approach for total energy and conformation similarity metrics. This work contributes to different areas ranging from evolutionary multi-objective optimization to bioinformatics as it extends the application universe of adaptive problem decomposition-based algorithms, which despite the success in various areas are practically unexplored in the PSP context.

Salivary detection of periodontopathic bacteria in Fanconi's anemia patients.

Fanconis anemia (FA) is characterized by bone marrow failure and can lead to infections such as periodontal disease. The aim of this study was to compare the prevalence of four periodontopathic bacteria including Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Fusobacterium nucleatum and Treponema denticola in saliva samples from children with and without FA. Paraffin-stimulated saliva samples were collected from 71 children and adolescents, aged 6-18 years old. The samples were divided in three groups: FA without HSCT (n = 25), FA with HSCT (n = 23) and Non-FA (n = 24). The test bacteria were identified using a 16S rRNA-based PCR analysis. P. gingivalis was the most frequent species in all groups and T. denticola the less frequent. P. gingivalis was also common species in combinations in all groups. No statistically difference was observed between the groups for single bacteria, pair or triple combination. The combination of the four species was detected in one saliva sample of the FA without HSCT group and in five samples of the Non-FA group, with statistical difference between them (p < 0.05, Fisher exact test). Systemic alterations found in FA subjects did not affect the salivary distribution of the four bacteria analyzed.

Tuning protein GlnB-Hs surface interaction with silicon: FTIR-ATR, AFM and XPS study.

Herbaspirillum seropedicae GlnB (GlnB-Hs) is a signal transduction protein involved in the control of nitrogen, carbon and energetic metabolism. The adsorption of GlnB-Hs deposited by spin coating on hydrophilic and hydrophobic silicon forms a thin layer that was characterized using atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared attenuated total reflectance spectroscopy (FTIR-ATR). AFM allowed the identification of globular, face-up donut like array of protein on hydrophilic silicon substrate, favoring deprotonated residues to contact the silicon oxide surface. Over hydrophobic silicon, GlnB-Hs adopts a side-on conformation forming a filament network, avoiding the contact of protonated residues with silicon surface. XPS allowed us to determine the protonated and non-protonated states of nitrogen 1s (N 1s). The FTIR-ATR measurements provided information about protein secondary structure and its conservation, after surface adsorption.

Regulation of Nitrogen Fixation in Herbaspirillum seropedicae

Herbaspirillum seropedicae is an aerobic, plant-associated, nitrogen-fixing bacterium of the β-proteobacteria group. Nitrogen fixation in this organism is repressed by ammonium ions or oxygen, and nitrogenase switch-off by ammonium ions is partial and does not involve ADP-ribosylation (Klasssen et al., unpublished). The nif genes of H. seropedicae are organized in at least two separate regions: region I contains contiguous nifA-and nifB-like genes (Souza et al., 1991a,b) and region II contains the nifHDK operon (Machado et al., 1996) contiguous to nifE, ORF4, modABCD and fixX. The glnA, ntrB and ntrC genes are contiguous and apparently constitute a single operon located away from the nif regions. Two glnB-like genes (Benelli et al., unpublished) and two rpoN genes are also present.

Streptococcus mutans GlnK protein: an unusual PII family member

Streptococcus mutans is a Gram-positive bacterium present in the oral cavity, and is considered to be one of the leading causes of dental caries. S. mutans has a glnK gene, which codes for a PII-like protein that is possibly involved in the integration of carbon, nitrogen and energy metabolism in several organisms. To characterize the GlnK protein of S. mutans, the glnK gene was amplified by PCR, and cloned into the expression vectors pET29a(+) and pET28b(+). The native GlnK-Sm was purified by anion exchange (Q-Sepharose) and affinity (Hi-Trap Heparin) chromatography. The GlnK-His-Sm protein was purified using a Hi-Trap Chelating-Ni2+ column. The molecular mass of the GlnK-His-Sm proteins was 85 kDa as determined by gel filtration, indicating that this protein is a hexamer in solution. The GlnK-His-Sm protein is not uridylylated by the Escherichia coli GlnD protein. The activities of the GlnK-Sm and GlnK-His-Sm proteins were assayed in E. coli constitutively expressing the Klebsiella pneumoniae nifLA operon. In K. pneumoniae, NifL inhibits NifA activity in the presence of high ammonium levels and the GlnK protein is required to reduce the inhibition of NifL in the presence of low ammonium levels. The GlnK-Sm protein was unable to reduce NifL inhibition of NifA protein. Surprisingly, the GlnK-His-Sm protein was able to partially reduce NifL inhibition of the NifA protein under nitrogen-limiting conditions, in a manner similar to the GlnK protein of E. coli. These results suggested that S. mutans GlnK is functionally different from E. coli PII proteins.